EAGER: Collaborative Research: Tracking of KOR1 Protein Transport in Arabidopsis using Fluorescent-Timer Imaging System

在这个项目中，将开发方法来直接观察蛋白质KORRIGAN1 （KOR1）在植物细胞中的运动。KOR1在纤维素纤维的形成中起作用，而纤维素纤维是植物细胞壁的主要成分。纤维素纤维沉积受KOR1控制，影响细胞生长和生物量生产。KOR1在细胞内从一个隔室移动到另一个隔室，最后到达细胞表面，纤维素纤维最终在那里沉积。为了可视化KOR1在植物细胞中的动态，本项目将应用荧光计时器（FT）技术，其中每个KOR1蛋白都标记有一个时间敏感的荧光标签。由于FT会随着时间的推移而改变其颜色，因此表达FT标记的KOR1的细胞的荧光图像将允许可视化KOR1在细胞内的年龄和位置。这将揭示KOR1在植物细胞中的运输途径。通过观察KOR1转运在不同生物环境下的变化，这些研究将揭示某些因素如何影响蛋白质在细胞内的运输路径。这是植物生物学中一种新颖的研究工具/方法，可以改变科学家对任何植物细胞中蛋白质的看法。在植物中，KOR1分布在不同的亚细胞结构域，如反式高尔基网络（TGN）、质膜（PM）、细胞质（TP）以及分裂细胞时的细胞板中，得到严格的维持。KOR1的功能障碍通常与TP靶向增加有关。该项目旨在可视化KOR1在端点位置分析之外的传输动态。将研究与KOR1生物学相关的两个转运过程。首先，我们将通过追踪KOR1在诱导蛋白表达后的年龄来分析de- nova合成的KOR1的运输路线/动力学和重定向机制。接下来，我们将利用KOR1年龄信息来剖析细胞分裂过程中KOR1到达细胞板的多重靶向途径。这些系统将作为进一步分析定量影响KOR1转运的各种因素的模型。跨学科团队将优化植物细胞生物学的FT技术，作为一个完整的荧光分析包，包括具有植物成熟时间特征的FT，兼容的荧光亚细胞标记和自动图像处理管道。本项目建立的实验平台将为进一步研究影响KOR1转运的多种因素奠定基础，包括蛋白质n -糖基化途径和与纤维素合酶复合物的相互作用。

In this project, methods will be developed to allow direct observation of the movement of a protein, KORRIGAN1 (KOR1), in plant cells. KOR1 functions in the formation of cellulose fibers, which are the primary constituents of plant cell walls. Cellulose fiber deposition that is controlled by KOR1 influences cell growth and biomass production. KOR1 travels from one compartment to another inside cells before it finally reaches the cell surface where cellulose fibers will ultimately be deposited. To visualize the dynamics of KOR1 in plant cells, this project will apply fluorescent-timer (FT) technology, in which each KOR1 protein is labeled with a time-sensitive fluorescent tag. Because FT changes its color over time, fluorescence images of cells expressing FT-tagged KOR1 will allow visualization of the age and location of KOR1 inside of the cells. This will reveal the route of KOR1 transport through the plant cell. By observing how KOR1 transport changes under different biological circumstances, these studies will reveal how certain factors influence the paths that proteins travel within cells. This is a novel research tool/approach in plant biology that can change the way the scientists view proteins in any plant cell.In plants, distribution of KOR1 among different subcellular domains, such as trans-Golgi network (TGN), plasmamembrane (PM), tonoplast (TP), as well as the cell plate in case of the dividing cells, is scrupulously maintained. Dysfunction of KOR1 is often associated with increased targeting to TP. This project aims to visualize the dynamics of KOR1 transport beyond analysis of endpoint locations. Two transport processes relevant to KOR1 biology will be studied. First, transport route/kinetics and a redirection mechanism of de-novo-synthesized KOR1 will be analyzed by tracking the age of KOR1 after induction of protein expression. Next, KOR1 age information will be used to dissect the multiple targeting routes of KOR1 to the cell plate during cell division. These systems will serve as models for further dissection of various factors that quantitatively affect KOR1 transport. The interdisciplinary team will optimize FT technology for plant cell biology as a whole fluorescence analysis package, including FT with characterized in planta maturation time, compatible fluorescent subcellular markers, and automated image-processing pipelines. The experimental platform established in this project will serve as the basis for further investigation of multiple factors that affect KOR1 transport, including protein N-glycosylation pathways and interactions with cellulose synthase complex.